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Induction of Gal Transcription from Repressor-Bound DNA (Tryptophan Fluorescence͞derepression͞galactose͞circular Dichroism)

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Induction of Gal Transcription from Repressor-Bound DNA (Tryptophan Fluorescence͞derepression͞galactose͞circular Dichroism) Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2957–2962, April 1997 Biochemistry Interaction of Gal repressor with inducer and operator: Induction of gal transcription from repressor-bound DNA (tryptophan fluorescenceyderepressionygalactoseycircular dichroism) SUMANA CHATTERJEE*, YAN-NING ZHOU†,SIDDHARTHA ROY*, AND SANKAR ADHYA†‡ *Department of Biophysics, Bose Institute, Calcutta 700 054, India, and †Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255 Contributed by Sankar Adhya, January 3, 1997 ABSTRACT Gal repressor inhibits transcription from view of the mechanism of induction does not apply to the P1 the gal promoter (P1) when it binds to the cognate operator promoter of the operon. (OE). The repression is relieved by the presence of the inducer The gal operon is transcribed by two promoters, P1 and P2 D-galactose. Compared with its interaction with free repres- (2). Repression of the two gal promoters involves Gal repressor sor, D-galactose binds to the repressor–operator complex with (GalR) binding to two operators, OE and OI (3–6). Derepres- 10-fold reduced affinity as determined by fluorescence en- sion occurs by the interaction of GalR with inducer D-galactose hancement measurements. Thermodynamic analysis and flu- or D-fucose (7). Whereas full repression of both P1 and P2 orescence anisotropy showed that the stability of the repres- requires the histone-like protein HU (8), interaction of re- sor–operator complex is reduced by only 7-fold by the presence pressor with OE alone represses P1, but not P2. GalRzOE- of the inducer in the complex. The formation of the inducer– mediated repression of P1 observed in the absence of HU also repressor–operator ternary complex has been confirmed by is relieved by the presence of D-galactose or D-fucose (6). We CD spectral analysis. Fluorescence spectroscopy and energy report here the results of an analysis of (i) formation and transfer experiments suggest that individual allosteric effects stability of various binary and ternary complexes of operator, of the two ligands, inducer and operator, on Gal repressor are repressor, and inducer by fluorescence and CD spectroscopy, responsible for the slightly weakened stability of the ternary (ii) the antagonistic effect of inducer or operator on the complex compared with the stability of the inducer–repressor binding of the other to repressor, and (iii) DNA-induced and repressor–operator complexes. In vitro transcription re- conformational changes in GalR. These results lead us to sults demonstrated full derepression of transcription of the P1 propose that inducer derepresses transcription in this system promoter under conditions in which the concentrations of the without dissociating the repressor from the operator. inducer–repressor binary complex are severalfold higher than the dissociation constant of the inducer–repressor–operator MATERIALS AND METHODS ternary complex into inducer–repressor and free DNA. These results strongly suggest that the inducer binding to the Materials. D-Galactose, D-fucose, adenosine 59-[g- repressor–operator complex does not lead to dissociation of thio]triphosphate (ATP[g-S]), and acrylamide were obtained the repressor from the operator during transcription induc- from Sigma. Fluorescein isothiocyanate and 5-({[(2- tion. Because Gal repressor inhibits transcription by modu- iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid (IAEDANS) were purchased from Molecular Probes. Oligo- lating the a subunit of the P1-bound RNA polymerase, we nucleotides purified by HPLC were from Midland Certified conclude that the inducer binding to the operator-bound Reagent (Midland, TX). The sequence of the 20-mer wild-type repressor only allosterically relieves the inhibitory effect of O was 59-TTGTGTAAACGATTCCACTA-39, whereas that repressor on RNA polymerase without dissociating the repres- E of the mutant O was 59-TTGTGTAAATAATTCCACTA-39. sor from DNA. E The sequence of the 29-mer wild-type OE was 59-xACTTCT- TGTGTAAACGATTCCACTAATTT-39, x being a hexyl- In the classical negative control of gene expression, a repressor amino group (2). Polynucleotide kinase was obtained from protein binds to cognate operator element(s) in DNA at or BRL. GalR was purified from a plasmid clone in which the near the promoter and inhibits transcription initiation (1). galR gene was expressed from a late promoter of bacteriophage Induction of transcription can occur in the presence of an T7 as described previously (9). The concentration of GalR inducer molecule that interacts with and causes an allosteric dimer was obtained from its extinction coefficient at 280 nm change in the repressor, which then loses its inhibitory power of 39,600 M21zcm21 (10) and is expressed in terms of mono- on transcription. It was originally proposed and commonly mer. Plasmid pSA509 used as the source of DNA in transcrip- believed that repressor sterically prevents RNA polymerase tion assays has been described before (6). from binding to the promoter site. According to this idea, IAEDANS Labeling of OE. Twenty nanomoles of OE DNA because of inducer-generated allosteric change(s), the repres- duplex, 250 mmol of ATP[g-S], and 10 units of T4 polynucle- sor loses its affinity for the operator and dissociates from otide kinase were incubated at 378Cfor2hin0.1Mpotassium DNA, thus allowing RNA polymerase to bind. We investigated phosphate buffer, pH 8.0. The mixture was extracted with an the dissociation of repressor from the operator DNA by equal volume of phenolychloroformyisoamyl alcohol inducer under conditions of transcription derepression in the (25:24:1). The aqueous layer was loaded onto a Sephadex G-25 gal operon of Escherichia coli and found that this long-held Abbreviations: IAEDANS, 5-({[(2-iodoacetyl)amino]ethyl}amino)- The publication costs of this article were defrayed in part by page charge naphthalene-1-sulfonic acid; AEDANS, 5-({[(acetyl)amino]ethyl}- payment. This article must therefore be hereby marked ‘‘advertisement’’ in amino)naphthalene-1-sulfonic acid; ATP[g-S], adenosine 59-[g-thio] accordance with 18 U.S.C. §1734 solely to indicate this fact. triphosphate. ‡To whom reprint requests should be addressed at: Developmental Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Genetics Section, Laboratory of Molecular Biology, Building 37, 0027-8424y97y942957-6$2.00y0 Room 2E16, 37 Convent Drive, MSC 4255, Bethesda, MD 20892- PNAS is available online at http:yywww.pnas.org. 4255. 2957 Downloaded by guest on October 7, 2021 2958 Biochemistry: Chatterjee et al. Proc. Natl. Acad. Sci. USA 94 (1997) column (1.5 3 12.5 cm), equilibrated with 0.1 M potassium «D and «A are the molar extinction coefficients of the donor phosphate (pH 8.0) and eluted. Appropriate fractions were and acceptor, respectively, at the excitation wavelength, and pooled, and IAEDANS was added at a final concentration of CD and CA are the concentrations of the donor and acceptor, 0.5 mM. The mixture was incubated for 90 min in the dark and respectively. The distance between the dansyl group and the extensively dialyzed. The calculated incorporation ratio ob- tryptophan in GalR was determined by the following equation: tained from A260 and A340 was approximately 1.2 mol per mol of oligonucleotide duplex. 21 1/6 R 5 Ro(E 2 1) , Synthesis and Purification of Fluorescein-Labeled OE. The 29-mer complementary oligonucleotides with a hexylamino group (x) attached at their 59 ends were separately synthesized where R is the distance between the donor and the acceptor, in an Applied Biosystems 381A DNA synthesizer using amino Ro is the distance for which the energy transfer efficiency is link supplied by Applied Biosystems. The oligonucleotides 50%, and E is the experimentally observed energy transfer were deprotected by incubating them at 558C for 24 h in 30% efficiency. ammonium hydroxide, precipitated by 1-butanol, then dried 2 24 1/6 3 under vacuum and dissolved in water. Ro 5 (Jk Qh ) (9.79 3 10 )cm, Ten milligrams of fluorescein 5-isothiocyanate was dissolved in 200 ml of 1.0 M NaHCO3yNa2CO3 buffer (pH 9.0)yN,N9- where J is the overlap integral (a measure of the spectral dimethylformamide water in the ratio of 5:2:3. The dye solu- y overlap), Q is the quantum yield, h is the refraction index, and tion, the oligonucleotide solution, and the carbonate buffer k is the orientation factor. The overlap integral was calculated were mixed in the ratio of 2:5:3, vortexed briefly, and incubated according to the method of Wu and Stryer (12). The value of at 258C for 20 h. The fluorescein-labeled oligonucleotides were k2 was taken as 2y3, which is completely spatially averaged. further purified by C18 reverse-phase HPLC using a linear gradient of 0–60% acetonitrile in 0.1 M triethylammonium Such complete spatial averaging is justified here because of acetate buffer (pH 7.0). The appropriate fractions of each aliphatic linkers separating the 5-({[(acetyl)amino]- labeled oligonucleotide were mixed in equimolar proportion, ethyl}amino)naphthalene-1-sulfonic acid (AEDANS) mole- and then annealed. cule from the DNA as well as the experimentally obtained low Fluorescence. Fluorescence spectra were recorded either in polarization value. The energy transfer experiments were done a Hitachi F3010 or SLM-Aminco (Urbana, IL) spectroflu- in the buffer described above at 258C. The excitation and orometer equipped with computers for spectra addition and emission band passes were 4 nm and 16 nm, respectively. The subtraction facility. Experiments were conducted in a water- emission wavelength was set at 500 nm. circulated thermostat chamber maintained at 258C. Excitation Circular Dichroism. CD spectra were obtained using a Jasco band pass was 1.5 nm, and emission band pass was 20 nm. J-600 spectropolarimeter at 258C. DNA concentration was 2.0 Samples were incubated at 258C for 30 min before conducting mM. Ten scans were averaged to produce a CD spectum.
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